U.S. patent number 6,916,656 [Application Number 10/487,235] was granted by the patent office on 2005-07-12 for non-linear amplitude dielectrophoresis waveform for cell fusion.
This patent grant is currently assigned to Cyto Pulse Sciences, Inc.. Invention is credited to Alan D. King, Derin C. Walters, Richard E. Walters.
United States Patent |
6,916,656 |
Walters , et al. |
July 12, 2005 |
Non-linear amplitude dielectrophoresis waveform for cell fusion
Abstract
An object of the invention is to provide a method of treating
biological cells prior to subjecting the biological cells to cell
fusion pulses which includes the step of treating the biological
cells with pre-fusion electric field waveforms which change
amplitude in a non-linear way with respect to time, such that the
biological cells are first aligned with a relatively low amplitude,
long duration pre-fusion electric field waveform and then
compressed with a relatively high amplitude, short duration
pre-fusion electric field waveform resulting in increased cell
membrane contact prior to being subjected to cell fusion. The
non-linear pre-fusion electric field waveforms can change in a
stepped way, in a continuous way, in a sigmoidal way, with
step-wise increasing waveforms in adjacent steps, with step-wise
increasing waveforms in non-adjacent steps, and in accordance with
non-linear algorithms.
Inventors: |
Walters; Richard E. (Columbia,
MD), Walters; Derin C. (Columbia, MD), King; Alan D.
(Highland, MD) |
Assignee: |
Cyto Pulse Sciences, Inc. (Glen
Burnie, MD)
|
Family
ID: |
23226725 |
Appl.
No.: |
10/487,235 |
Filed: |
October 25, 2004 |
PCT
Filed: |
April 05, 2002 |
PCT No.: |
PCT/US02/08239 |
371(c)(1),(2),(4) Date: |
October 25, 2004 |
PCT
Pub. No.: |
WO03/02091 |
PCT
Pub. Date: |
March 13, 2003 |
Current U.S.
Class: |
435/450; 435/461;
435/470; 435/471 |
Current CPC
Class: |
C12M
35/02 (20130101); C12N 5/16 (20130101); C12N
13/00 (20130101); A61K 2039/5152 (20130101); A61K
2039/5154 (20130101); H01L 2224/83856 (20130101); H01L
2924/01019 (20130101) |
Current International
Class: |
C12N
15/02 (20060101); C12N 15/64 (20060101); C12N
15/87 (20060101); C12N 15/74 (20060101); C12N
015/02 (); C12N 015/64 () |
Field of
Search: |
;435/450,461,470,471 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Ketter; James
Attorney, Agent or Firm: Townsend; Marvin S.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATION
This application claims priority based upon copending U.S.
Provisional Application Ser. No. 60/315,936, filed 31 Aug. 2001.
Claims
What is claimed is:
1. A method of fusing or electroporating biological cells
comprising the steps of: treating the biological cells with a
pre-fusion electric field waveform which changes amplitude in a
non-linear way with respect to time, and followed by subjecting the
biological cells to a fusion or electroporation pulse.
2. The method of claim 1 wherein the biological cells are first
aligned and then compressed resulting in increased cell membrane
contact prior to being subjected to cell fusion.
3. The method of claim 1 wherein the pre-fusion electric field
waveform amplitude includes a relatively low amplitude, long
duration electric field waveform followed by a relatively high
amplitude, short duration electric field waveform.
4. The method of claim 1 wherein the pre-fusion electric field
waveform amplitude changes in a stepped non-linear way with respect
to time.
5. The method of claim 1 wherein the pre-fusion electric field
waveform amplitude changes in a continuous non-linear way with
respect to time.
6. The method of claim 1 wherein the pre-fusion electric field
waveform includes an AC electric field waveform which changes
amplitude in a non-linear way with respect to time.
7. The method at claim 6 wherein the amplitude of said AC electric
field waveform changes in a non-linear way with respect to time in
accordance with a non-linear algorithm.
8. The method of claim 6 wherein said AC electric field waveform
has an AC-waveform electric field intensity between 10 volts/cm and
1,000 volts/cm.
9. The method of claim 1 wherein the pre-fusion electric field
waveform amplitude includes non-linear step-wise increasing
waveforms applied as pre-fusion electric field waveforms, and
wherein the waveforms are provided as either adjacent steps or
non-adjacent steps.
10. The method of claim 1, further including the steps of: treating
the biological cells with an AC electric field waveform following
the cell fusion or electroporation pulse.
11. A method of treating biological cells prior to subjecting the
biological cells to cell fusion, comprising the step of: treating
the biological cells with an electric field amplitude which changes
in a non-linear way with respect to time, such that the biological
cells are aligned and have increased cell membrane contact, and
such that the biological cells are compressed against one another
prior to being subjected to cell fusion.
12. The method of claim 1, further comprising the step of
subjecting the biological cells to a post-fusion or
post-electroporation electric field waveform.
Description
TECHNICAL FIELD
The present invention relates generally to methods and apparatus
for fusing biological cells to one another. More specifically, the
present invention provides methods and apparatus for treating
biological cells with electrical fields, such that the biological
cells are aligned and have increased cell membrane contact prior to
being subjected to cell fusion.
BACKGROUND ART
If a neutrally charged biological cell is placed in a uniform
electric field, such as provided by a pair of electrodes which are
both planar, the biological cell does not move toward one electrode
or another because the attractive forces from both electrodes are
the same.
On the other hand, if a neutrally charged biological cell is placed
in a non-uniform electric field, such as provided by two electrodes
which are both not planar, as shown in PRIOR ART FIG. 1, the
biological cell forms a dipole, is attracted to one electrode with
greater attractive force than the other, and moves towards the
electrode having the greater attractive force.
Such a use of a non-uniform electric field is used in
dielectrophoresis, and the concept of using dielectrophoresis to
align living cells, followed by a fusion/electroporation pulse, to
fuse cells has been in the literature since early 1970's. This
process is used to produce hybrids of two different cell types for
therapeutic purposes, for hybridoma production for producing
monoclonal antibodies, for nuclear fusion, and for producing other
hybrid cells. Dielectrophoresis is the process of applying an
electrical force on neutrally charged particles such as living
cells. The force from dielectrophoresis results from applying a
non-uniform electric field that separates charges inside the cells
forming a dipole. After the dipole has been formed, the non-uniform
electric field then moves the cells towards the highest or lowest
electric field intensity. This movement is dependent on the
relative conductivities and permittivities of the medium and the
biological cells or particles. The dielectrophoretic force is a
function of the electric field squared so electric field polarity
is not important. The force is a function of the relative
conductivities and permitivities of the medium and the particles or
cells. The conductivities and permitivities are also a function of
frequency of the applied electric field. Typically, an AC voltage
wave, such as a sine wave, is applied across electrodes to produce
this alternating electric field. The sine wave voltage, frequency,
and duration are optimized for specific cell types. After the AC
wave is applied to align the cells, one or more
fusion/electroporation pulses are applied to form pathways in the
cell membranes in which membranes from both cells commingle. These
pathways permit the contents of the cells to mix forming a hybrid
cell. Following the fusion pulses, another AC field can be applied
to hold the cells together while the fused cells stabilize. In some
cases, the AC voltage has been linearly increased or decreased to
prevent damage to the cells due to a sudden application of a
field.
Examples of cell fusion applications include hybridoma production
and nuclear transfer. A recent application for electrofusion is to
produce therapeutic hybrids for cancer immunotherapy. These hybrids
are produced from cancer tumor cells and immune system dendritic
cells in an ex vivo process. Each treatment requires a large number
of viable hybrids, which results in a new requirement for high
efficiency in the hybrid production process.
There are a number of techniques (electrical, mechanical, chemical)
available to perform cell fusion. This invention relates to
electrical means. The current electric art uses a voltage waveform
generator connected to an electrode device. With respect to
relevant known electrical, mechanical, and chemical techniques, the
following U.S. Patents and published PCT application are of
particular interest and are incorporated herein by reference:
4,326,934 Apr. 27, 1982 Pohl 4,441,972 Apr. 10, 1982 Pohl 4,764,473
Aug. 16, 1988 Matschke et al 4,784,954 Nov. 15, 1988 Zimmermann
5,304,486 Apr. 19, 1994 Chang 6,010,613 Jan. 4, 2000 Walters et al
WO 00/60065 Oct. 12, 2000 Walters et al
From the above, it is known to use pre-fusion electric field
waveforms that have either a constant amplitude, see PRIOR ART FIG.
3, or a linearly increasing amplitude, see PRIOR ART FIG. 4. FIG. 5
illustrates an overall general PRIOR ART protocol for carrying out
cell fusion using electric field waveforms, wherein a pre-fusion
electric field waveform is followed by a fusion/electroporation
pulse, which is followed by a post-fusion electric field
waveform.
Nevertheless, efficiency of cell fusion following a constant
amplitude or a linearly increasing amplitude of pre-fusion electric
field waveforms cannot deliver the higher efficiencies required in
such applications as therapeutic hybrid production for cancer
immunotherapy. In this respect, it would be desirable if pre-fusion
electric field waveforms were provided for biological cells which
increases cell fusion efficiency over biological cells treated with
a constant amplitude or a linearly increasing amplitude pre-fusion
electric field waveform.
More specifically with respect to U.S. Pat. No. 5,304,486 of Chang,
it is noted that FIG. 2E of Chang discloses a linear low voltage
presine AC waveform, a high voltage linear electroporating AC
waveform, and a low voltage linear post-poration AC waveform. The
invention of Chang is confined solely to the fusion/electroporation
pulses. Chang discloses only a linear, low voltage presine AC
waveform. Chang does not disclose a non-linear low voltage presine
AC waveform. Chang does not focus attention on the presine AC
waveform, other than a nominal statement thereof.
The first process in any cell fusion system is to bring the cells
into contact. In any case, sufficient force must be applied to each
cell to overcome the negative surface charge. Merely applying a
uniform electric field will not move a cell because the net charge
of the cell is zero. Thus from the definition of electric field,
there is no force applied:
However, a non-uniform field moves the positive ions inside each
cell to one side and the negative ions to the opposite side
producing a dipole, as shown in PRIOR ART FIG. 1. Once the dipole
is induced, a net force is exerted, on the cell because the
intensity of the field is greater on one side than the other. The
movement of cells in one direction causes the cells to concentrate
in an area. Since the cells are now dipoles, the negative side of
one cell will attract the positive side of another cell overcoming
the negative surface charge, as shown in PRIOR ART FIG. 2. The
non-uniform electric field is produced by the electrode device. The
non-uniformity is a function of the electrode configuration, as
shown in PRIOR ART FIGS. 1 and 2.
Generally, the cell types to be fused are placed in a low
conductive medium (less than 0.01 S/m) to minimize ohmic heating
that may harm the cells and that causes turbulence thus reducing
the number of fused hybrids. In this respect, it would be desirable
for biological cells being subjected cell fusion to be treated so
as to reduce heating during cell alignment and cell membrane
contact.
The waveform generator has two functions. The first is to produce
the AC voltage waveform that is converted into an AC field by the
electrode device. This AC field then brings the cells into
alignment/contact. The second function is to produce a pulse
voltage that electroporates the cell membrane, fusing the cells. In
some cases another AC voltage is produced after the fusing pulse to
hold the cells in alignment until the fusion products become viable
or stable.
One of the factors for successful fusion is the membrane contact
between the adjacent cells. The closer this contact before the
fusion pulse is applied, the higher the efficiency of fusion. In U.
Zimmermann, et al., "Electric Field Induced Cell to Cell Fusion",
J. Membrane Biol. 67, 165-182 (1982), Zimmermann points out that
increasing the AC wave electric field strength just before the
fusion pulse may be the optimum approach. Clearly, it would be
desirable for biological cells that are to undergo cell fusion to
be pretreated with pre-fusion electric field waveforms which bring
abort increased cell membrane contact without turbulence or
heating.
In addition, there are a number of reasons why it is not desirable
to immediately provide a high amplitude alignment waveform to cells
that are to undergo cell fusion. A first reason is a mechanical
reason. That is, immediate application of a high amplitude
alignment waveform causes extreme force to be exerted on the cells,
causing the cells to move rapidly towards an electrode. This rapid
cell movement causes turbulence forces in the medium surrounding
the cells. The turbulence forces do not allow complete pearl chains
of cells to form, and the turbulence forces cause already formed
pearl chains of cells to break up.
A second reason why it is not desirable to immediately provide a
high amplitude alignment waveform to cells that are to undergo cell
fusion is that such a high amplitude alignment waveform causes
heating to occur in the media in which the biological cells are
suspended. Heating also causes turbulence which does not permit
complete pearl chains of aligned cells to form and causes already
formed pearl chains to break up. The heat in the heated up media
also reduces cell viability.
In view of the above, it would be desirable to avoid the mechanical
forces, turbulence, and heating which result from immediately
applying a high amplitude alignment waveform to biological cells
that are to undergo cell fusion.
Thus, while the foregoing body of prior art indicates it to be well
known to use pre-fusion electric field waveforms prior to carrying
out cell fusion with en electroporation pulse, the prior art
described above does not teach or suggest a dielectrophoresis
waveform for cell fusion which has the following combination of
desirable features: (1) provides pre-fusion electric field
waveforms for biological cells which increase cell fusion
efficiency over biological cells treated with a constant amplitude
or a linearly increasing amplitude pre-fusion electric field
waveforms; (2) avoids the mechanical forces, turbulence, and
heating which result from immediately applying a high amplitude
alignment waveform to biological cells that are to undergo cell
fusion; (3) reduces heating of biological cells being treated with
pre-fusion electric field waveforms for increasing cell alignment
and cell membrane contact prior to being subjected to cell fusion;
and (4) increase cell membrane contact between biological cells
treated with pre-fusion electric field waveforms prior to
undergoing cell fusion. The foregoing desired characteristics are
provided by the unique non-linear dielectrophoresis waveform for
cell fusion of the present invention as will be made apparent from
the following description thereof. Other advantages of the present
invention over the prior art also will be rendered evident.
Additional U.S. patents that are of interest include:
4,561,961 Dec. 31, 1985 Hofmann 5,001,056 Mar. 19, 1991 Snyder et
al 5,589,047 Dec. 31, 1996 Coster et al 5,650,305 Jul. 22, 1997 Hui
et al
Additional literature references include: 1. R. Bischoff, et al.,
"Human Hybridoma Cells Produced by Electro-Fusion", Fed. Eur.
Biochem. Soc. Lett. 147, 64-68 (1982). 2. L. Changben, et al., "Use
of Human Erythrocyte Ghosts for Transfer of 125.sub.I-BSA and
125.sub.I-DNA into Animal Cells from Cell Fusion", Scientia Sinica
(Series B) 25, 680-865 (1982). 3. C. S. Chen, et al., "Biological
Dielectrophoresis: The Behavior of Lone Cells in a Non-uniform
Electric Field", Ann. N.Y. Acad. Sci. 238, 176-185 (1974). 4.
Coster, H. G. L. and Zimmermann, U. "Direct Demonstration of
Dielectric Breakdown in the Membranes of Valonia utricularis. "
Zeitschrift fur Naturforschung. 30 c, 77-79.1975. 5. Coster, H. G.
L. and Zimmermann, U. "Dielectric Breakdown in the Membranes of
Valonia utricularis: the role of energy dissipation". Biochimica et
Biophysica Acta. 382, 410-418, 1975. 6. Coster, H. G. L. and
Zimmermann, U. "The mechanisms of Electrical Breakdown in the
Membranes of Valonia utricularis." Journal of Membrane Biology. 22,
73-90, 1975. 7. K. Kaler, et al., "Dynamic Dielectrophoretic
Levitation of Living Individual Cells", J. Biol. Phys. 8, 18-31
(1980). 8. A. R. Murch; et al., "Direct Evidence that Inflammatory
Multi-Nucleate Giant Cells Form by Fusion", Pathol. Soc. Gr. Brit.
Ire. 137, 177-180 (1982). 9. Neumann, Bet al. "Cell Fusion Induced
by High Electrical Impulses Applied to Dictyostelium",
Naturwissenschaften 67, 414, 1980 10. Petrucci, General Chemistry:
Principles and Modern Applications, 4th ed., p. 621, 1985 (no
month). 11. Zimmermann et al., Electric Field-Induced Cell-to-Cell
Fusion, The Journal of Membrane Biology, vol. 67, pp. 165-182
(1982) [no month]. 12. Pohl, H. "Dielectrophoresis", Cambridge
University Press, 1978. 13. H. A. Pohl, "Biophysical Aspects of
Dielectrophoresis", J. Biol. Phys. 1, 1-16 (1973). 14. H. A. Pohl,
et al., "Continuous Dielectrophoretic Separation of Cell Mixtures",
Cell Biophys. 1, 15-28 (1979). 15. H. A. Pohl, et al.,
"Dielectrophoretic Force", J. Biol. Phys. 6, 133 (1978). 16. H. A.
Pohl, et al., "The Continuous Positive and Negative
Dielectrophoresis of Microorganisms", J. Bio. Phys. 9, 67-86
(1981). 17. Sale, J. H. and Hamilton, W. A. "Effects of High
Electric Fields on Micro-Organisms", Biochimica et Biophysica Acta.
163, 37-43, 1968. 18. Sepersu, E. H., Kinosita, K. and Tsong, T. Y.
"Reversible and Irreversible Modification of Erythrocyte Membrane
Permeability by Electric Fields" Biochimica et Biophysica Acta.
812, 779-785, 1985. 19. J. Vienken, et al., "Electric Field-Induced
Fusion: Electro-Hydraulic Procedure for Production of Heterokaryon
Cells in High Yield", Fed. Eur. Biomed. Soc. Lett. 137, 11-13
(1982). 20. H. Weber, et al., "Enhancement of Yeast Protoplast
Fusion by Electric Field Effects", A Preprint for Proceedings of
the Fifth International Symposium on Yeasts, London, Ontario,
Canada, Jul. 80. 21. Zimmermann, U. "Electrical Field Mediated
Fusion and Related Electrical Phenomena", Biochimica et Biophysica
Acta. 694, 227-277. 1982. 22. Zimmermann, U. et al "Fusion of Avena
Sativa Mesophyll Proptoplasts by Electrical Breakdown", Biochimica
et Biophysica Acta. 641, 160-165, 1981, 1982. 23. U. Zimmermann, et
al., "Electric Field-Induced Release of Chloroplasts from Plant
Protoplasts", Naturwissen 69, 451 (1982). 24. U. Zimmermann, et
al., "Electric Field-Mediated Cell Fusion", J. Biol. Phys. 10,
43-50 (1982). 25. U. Zimmermann, "Cells with Manipulated Functions:
New Perspectives for Cell Biology, Medicine, and Technology",
Angew. Chem. Int. Ed. Engl. 20, 325-344 (1981).
DISCLOSURE OF INVENTION
The present invention is an improvement over the current art. With
the present invention, a non-linear voltage waveform having a
non-linear change in amplitude is applied to biological cells
before application of one or more cell fusion/electroporation
pulses. The present invention first brings about or forces
tangential membrane contact and alignment between adjacent cells as
a result of applying the non-linear voltage waveform. Then, the
present invention brings about or forces close membrane contact
between adjacent cells as a result of further application of the
non-linear voltage waveform. In this respect, the biological cells
10 are compressed against each other under the influence of the
waveform with a non-linear change in amplitude. The biological
cells can be similar Eukaryotic cells, or they can be dissimilar
Eukaryotic cells.
The pre-fusion non-linear voltage waveform, which changes in
amplitude in a non-linear way, can be a non-linear AC voltage
waveform. The AC electric field waveforms can include sine waves. A
parameter of the non-linearity of the change in amplitude of the
waveform can be set so that the fusion process can be optimized by
cell type. With the amplitude of the AC waveform varying
non-linearly in amplitude over time, the biological cells align and
fuse with lower energy (less heating) and with higher fusion
efficiency.
Preferably, the pre-fusion electric field waveform includes a
relatively low amplitude, long duration electric field waveform
followed by a relatively short duration, high amplitude electric
field waveform. More specifically, the relatively low amplitude,
long duration electric field waveform slowly facilitates pearl
chain formation and alignment of biological cells without causing
turbulence or cell death. Once the cells are aligned and in pearl
chains, a relatively high amplitude, short duration pre-fusion
electric field waveform is applied to the biological cells. The
cells are already in alignment, and for a short period of time,
before heating occurs, cell compression takes place without
turbulence.
The pre-fusion electric field waveform amplitude can change in a
stepped non-linear way with respect to time. The pre-fusion
electric field waveform can change in amplitude in a continuous
non-linear way with respect to time.
The pre-fusion electric field waveform includes an AC electric
field waveform which changes in amplitude in a non-linear way with
respect to time. The amplitude of the AC electric field waveform
can change in a non-linear way with respect to time in accordance
with a non-linear algorithm.
Preferably, the AC electric field waveforms have an AC waveform
electric field intensity between 10 volts/cm and 1,000
volts/cm.
The non-linear step-wise amplitude increasing waveforms can be
applied as pre-fusion electric field waveforms in either adjacent
steps or non-adjacent steps.
Subsequent to applying the pre-fusion electric field waveform which
changes in amplitude in a non-linear way, the biological cells are
subjected to a cell fusion pulse. In addition, the biological cells
can be treated with a non-linear AC electric field waveform
following the cell fusion pulse.
There are a number of embodiments of a non-linear waveform whose
amplitude changes in a non-linear way with respect to time such as
wherein a pre-fusion type of AC amplitude changes with time.
Examples of algorithms that can be used for the non-linear change
in amplitude over time include exponential, logarithmic,
polynomial, power function, step function, sigmoid function, and
non-linear algorithms generally, etc.
In view of the above, an object of the present invention is to
provide a new and improved non-linear pre-fusion electric field
waveform whose amplitude changes in a non-linear way with respect
to time as a dielectrophoresis waveform prior to cell fusion.
Another object of the invention is to provide a non-linear
dielectrophoresis waveform for cell fusion in which AC electric
field waveforms applied to the biological cells increase cell
fusion efficiency over biological cells treated with a constant
amplitude or a linearly increasing amplitude pre-fusion electric
field waveform.
Yet another object of the present invention is to provide a new and
improved non-linear dielectrophoresis waveform for cell fusion
which reduces heating of biological cells being treated with
pre-fusion electric field waveforms, for increasing cell alignment
and cell membrane contact prior to being subjected to cell
fusion.
Still another object of the present invention is to provide a new
and improved non-linear dielectrophoresis waveform for cell fusion
that avoids the mechanical forces, turbulence, and heating which
result when the biological cells are subjected to a cell fusion
from immediately applying a high amplitude alignment waveform to
biological cells that are to undergo cell fusion.
Even another object of the present invention is to provide a new
and improved non-linear dielectrophoresis waveform for cell fusion
that increases cell membrane contact between biological cells
treated with pre-fusion electric field waveforms prior to
undergoing cell fusion.
These together with still other objects of the invention, along
with the various features of novelty which characterize the
invention, are pointed out with particularity in the claims annexed
to and forming a part of this disclosure. For a better
understanding of the invention, its operating advantages and the
specific objects attained by its uses, reference should be had to
the accompanying drawings and descriptive matter in which there are
illustrated preferred embodiments of the invention.
BRIEF DESCRIPTION OF DRAWINGS
The invention will be better understood and the above objects as
well as objects other than those set forth above will become more
apparent after a study of the following detailed description
thereof. Such description makes reference to the annexed drawing
wherein:
FIG. 1 illustrates PRIOR ART dipole formation in biological cells
under the influence of a non-uniform electric field created by
non-symmetrical electrodes.
FIG. 2 illustrates a PRIOR ART path of movement of a biological
cell in a non-uniform electric field created by non-symmetrical
electrodes and also illustrates pearl chain alignment and formation
of biological cells.
FIG. 3 illustrates PRIOR ART a constant amplitude pre-fusion
electric field waveform.
FIG. 4 illustrates PRIOR ART a linearly increasing amplitude
pre-fusion electric field waveform.
FIG. 5 illustrates an overall general PRIOR ART protocol for
carrying cut cell fusion using electric field waveforms, wherein a
pre-fusion electric field waveform is followed by a
fusion/electroporation pulse, which is followed by a post-fusion
electric field waveform.
FIG. 6 shows independent biological cells prior to applying
non-linear dielectrophoresis waveforms of the invention.
FIG. 7 shows tangentially contacting biological cells in pearl
chain alignment during application of a relatively low amplitude,
long duration pre-fusion electric field waveform of the
invention.
FIG. 8 shows closely contacting and compressed biological cells
during application of a relatively high amplitude, short duration
pre-fusion electric field waveform of the invention, following the
application of the relatively low amplitude, long duration
pre-fusion electric field waveform that was applied in FIG. 7.
FIG. 9 shows variations in pre-fusion electric field waveforms
applied to biological cells using a power function having
variations in the constant "k" of the, power function. It is noted
that for each selection of the constant "k", there is a relatively
low amplitude, long duration pre-fusion electric field waveform
portion followed by a relatively high amplitude, short duration
pre-fusion electric field waveform portion.
FIG. 10 shows a selected "k" modulated non-linear increasing
continuous AC waveform applied as a pre-fusion electric field AC
waveform as a power function with a selected power function
constant "k" shown in FIG. 9, a relatively low amplitude, long
duration pre-fusion electric field waveform portion is shown
followed by a relatively high amplitude, short duration pre-fusion
electric field waveform portion.
FIG. 11 shows non-linear sigmoidally shaped waveforms applied as
pre-fusion electric field waveforms, wherein a transition from a
relatively low amplitude, long duration pre-fusion electric field
waveform to a relatively high amplitude, short duration pre-fusion
electric field waveform is relatively slow.
FIG. 12 shows non-linear sigmoidally shaped waveforms applied as
pre-fusion electric field waveforms, wherein a transition from a
relatively low amplitude, long duration pre-fusion electric field
waveform to a relatively high amplitude, short duration pre-fusion
electric field waveform is relatively fast.
FIG. 13 shows non-linear step-wise increasing waveforms applied as
pre-fusion electric field waveforms, wherein the pre-fusion
electric field waveforms are provided as non-adjacent steps,
wherein a first pre-fusion electric field waveform is a relatively
low amplitude, long duration pre-fusion electric field waveform,
wherein an off-time is provided, and wherein a second pre-fusion
electric field waveform is a relatively high amplitude, short
duration pre-fusion electric field-waveform.
FIG. 14 shows non-linear step-wise increasing waveforms applied as
pre-fusion electric field waveforms, wherein the pre-fusion
electric field waveforms are provided as adjacent steps, wherein a
first pre-fusion electric field waveform is a relatively low
amplitude, long duration pre-fusion electric field waveform, and
wherein a second pre-fusion electric field waveform is a relatively
high amplitude, short duration pre-fusion electric field waveform
and is applied immediately after the first pre-fusion electric
field waveform.
MODES FOR CARRYING OUT THE INVENTION
A method and apparatus are provided for non-linear
dielectrophoresis waveform for cell fusion, and with reference to
the drawings, said method and apparatus are described below.
The present invention is an improvement over the current art. With
the present invention, a non-linear voltage waveform, whose
amplitude changes in a non-linear way, is applied to biological
cells before application of one or more cell fusion pulses.
Separated biological cells 10 are shown in FIG. 6. The present
invention first brings about or forces tangential membrane contact
between adjacent cells as a result of applying the non-linear
voltage waveform, as shown in FIG. 7. Then, the present invention
brings about or forces close membrane contact between adjacent
cells as a result of applying the non-linear voltage waveform, as
shown in FIG. 8. As shown in FIG. 8, the biological cells 10 are
compressed against each other under the influence of the non-linear
voltage waveform.
The non-linear voltage waveform, whose amplitude changes in a
non-linear way, can be a non-linear AC voltage waveform. A
parameter of the non-linearity of the waveform can be set so that
the fusion process can be optimized by cell type. With the
amplitude of the AC waveform varying non-linearly over time, the
biological cells align and fuse with lower energy (less heating)
and with higher fusion efficiency.
More specifically, with reference to FIG. 10, with a non-linear AC
voltage waveform, preferably the non-linear AC voltage waveform has
a relatively low AC voltage amplitude at the first portion of the
waveform that brings the biological cells into close proximity and
alignment. A second portion of the waveform then increases in
amplitude just before the fusion pulse is to be applied. This
increase in amplitude produces a short-term, intense, and
non-uniform electric field, which forces the biological cells into
close contact. Heating is reduced, due to the lower voltage used
for alignment and the shorter duration intense AC portion.
There are a number of embodiments of this type of AC amplitude
change with time, for example, exponential, logarithmic,
polynomial, power function, step function, sigmoid function, and
non-linear algorithm generally, etc.
One embodiment of this non-linear waveform is a power function that
may be represented by the following mathematical formula or
algorithm.
The amplitude of the AC waveform as a function of time=
The total AC waveform duration, the AC starting amplitude, the AC
stopping amplitude and the power exponent "k" are all optimized for
the cells type being used.
The effect of varying the power exponent "k" is illustrated by the
graphs in FIG. 9. A particular power function graph is illustrated
in FIG. 10 where "k" equals 2. Thus, the invention provides a
relatively low amplitude, long duration pre-fusion electric field
waveform that produces a lower intensity electric field to align
the biological cells and that then provides a relatively high
amplitude, short duration pre-fusion electric field waveform of
increased electric field intensity to force the cells into close
contact, just before the AC wave ends and the cell
fusion/electroporation pulses begins. This non-linear change in
amplitude approach of the invention also produces less heating and
less turbulence, which further provide an increase in cell hybrid
production and production efficiency.
As stated above, in FIG. 10 there is a showing of non-linear,
amplitude increasing continuous waveforms applied as pre-fusion
electric field waveforms as a power function with a selected power
function constant of "k" equals 2.
FIG. 11 shows non-linear sigmoidally shaped waveforms, whose
amplitudes change in a non-linear way, applied as pre-fusion
electric field waveforms, wherein a transition from a relatively
low amplitude, long duration pre-fusion electric field waveform to
a relatively high amplitude, short duration pre-fusion electric
field waveform is relatively slow.
FIG. 12 shows non-linear sigmoidally shaped waveforms, whose
amplitudes change in a non-linear way, applied as pre-fusion
electric field waveforms, wherein a transition from a relatively
low amplitude, long duration pre-fusion electric field waveform to
a relatively high amplitude, short duration pre-fusion electric
field waveform is relatively fast.
FIG. 13 shows non-linear step-wise increasing waveforms, whose
amplitudes change in a non-linear way, applied as pre-fusion
electric field waveforms, wherein the pre-fusion electric field
waveforms are provided as non-adjacent steps, wherein a first
pre-fusion electric field waveform is a relatively low amplitude,
long duration pre-fusion electric field waveform, wherein an
off-time is provided, and wherein a second pre-fusion, electric
field waveform is a relatively high amplitude, short duration
pre-fusion electric field waveform.
FIG. 14 shows non-linear step-wise increasing waveforms, whose
amplitudes change in a non-linear way, applied as pre-fusion
electric field waveforms, wherein the pre-fusion electric field
waveforms are provided as adjacent steps, wherein a first
pre-fusion electric field waveform is a relatively low amplitude,
long duration pre-fusion electric field waveform, and wherein a
second pre-fusion electric field waveform is a relatively high
amplitude, short duration pre-fusion electric field waveform and is
applied immediately after the first pre-fusion electric field
waveform.
The present invention can be carried out by an apparatus that
delivers such pre-fusion electric field waveforms described above.
In this respect, a software modification has been made to the Cyto
Pulse PA-4000 system with the PA-101 AC waveform generator. This
Cyto Pulse Sciences, Inc. PulseAgile (Reg. U.S. Pat. and Tm. Off.)
system software now produces this waveform (see U.S. Pat. No.
6,010,613 incorporated herein by reference). The non-linear AC
waveform parameters are inputted by the user, and the computer
generates the AC waveforms (pre-fusion electric field waveforms)
and fusion pulse waveforms (electroporation waveforms).
Experiments were carried out, and improvements of cell: fusion were
recorded using stepped pre-fusion electric field waveforms whose
amplitudes change in a non-linear way.
A549 cells were purchased from the ATCC. The cells were thawed and
placed in tissue culture flasks with medium recommended by ATCC.
Dendritic cells prepared by culture of peripheral blood mononuclear
cells in a mixture of cytokines for 7 days were used as fusion
partners.
8 million cells/milliliter of each cell, type were, mixed equally
to yield a final concentration of 4 million cells/milliliter of
each cell type in the mixture. Cell suspension volumes of 3
milliliters were used for each fusion.
The procedure consisted of the following steps. Cells were
centrifuged and re-suspended in 10 milliliters of Cyto Pulse
commercial Cytofusion medium (formula C) The cells were washed
twice in the same medium and re-suspended in Cytofusion medium
after the washes. Cells were counted and the cell concentration was
adjusted to 8 million cells/milliliter. Equal volumes of A549 cells
and dendritic cells were mixed. Three milliliters of cell
suspension was placed into a 6 milliliter capacity coaxial cell
fusion electrode, having an internal cylindrical anode of 3.9 cm
diameter with a gap of 4 mm from the cathode. The following cell
fusion protocols were applied.
The parameters for the two experimental and one control groups are
as follows:
Group A: (Invention)
First pre-fusion electric field waveform: 45 V to 45 V, 20 Seconds,
0.8 MHz
Second pre-fusion electric field waveform: 75 V to 75 V. 10
seconds, 0.8 MHz
Fusion/electroporation pulse 1.times.800 V, 40 microseconds
Post fusion/electroporation pulse 45 V to 45 V, 50 seconds, 0.8
MHz
Group B: (Prior Art)
First pre-fusion electric field waveform: 75 V to 75 V. 10 seconds,
0.8 MHz
Fusion/electroporation pulse 1.times.800 V, 40 microseconds
Post fusion/electroporation pulse 45 V to 45 V, 50 seconds, 0.8
MHz
Group C: (Control--No Electricity)
After fusion, cells were left undisturbed for 30 minutes to allow
fusion maturation. Three milliliters of tissue culture medium with
10% fetal bovine serum were added to the cell suspension in the
cell fusion electrode. Fifteen minutes later the cells were
harvested for analysis.
An aliquot of cells was placed onto a silinized microscope slide
using a commercial Cytospin (Shandon) centrifuge. The cells were
identified using immunohistochemistry. A549 cells were identified
using anti-keratin monoclonal antibodies and the dendritic cells
were identified by using anti human HLA-DR monoclonal antibodies.
Meyers hematoxylain was used for a nuclear counterstain. The cells
with either brown keratin staining or red HLA-DR staining or both
were manually counted.
Results are shown in the TABLE below in which percentages of fused
cells are presented for mixed A549 and dendritic cells in Group A
(which were treated with a pre-fusion electric field waveform of
the invention prior to cell fusion), for mixed A549 and dendritic
cells in Group B (which were treated by a prior art pre-fusion
electric field waveform prior to cell fusion), and for mixed A549
and dendritic cells in Group C (which were pot treated by any
pre-fusion electric field waveform at all prior to cell
fusion).
TABLE Fused Fused Fused Group A549/A549 den./den. den./A549 A
(Invention) 7.2% 5.4% 22.7% B (Pr.Art) 6.8% 4.0% 18.1% C (Control)
2.2% 2.2% 10.8%
By way of explanation of the TABLE, the first column lists the
respective groups of A549 and dendritic cells (den.) that were
subjected to fusion treatment. The second column lists the
percentages of fused cells formed by the fusion of an A549 cell
with another A549 cell. The third column lists percentages of fused
cells formed by the fusion of a dendritic cell with another
dendritic cell. The fourth column lists percentages of fused cells
formed by the fusion of a dendritic cell with an A549 cell.
It is noted that for each type of cell fusion (A549/A549,
den./den., and den./A549), the percentages of fused cells are
greater with Group A (employing a pre-fusion electric field
waveform of the invention) than with either Group B (employing a
pre-fusion electric field waveform of the prior art) or Group C
(employing no pre-fusion electric field waveform at all). More
specifically, 7.2% is greater than 6.8%, which is greater than
2.2%. Also, 5.4% is greater than 4.0%, which is greater than 2.2%.
Also, 22.7% is greater than 18.1%, which is greater than 10.8%. In
summary, employing a pre-fusion electric field waveform of the
invention provides higher fusion efficiency than employing either a
prior art pre-fusion electric field waveform or no pre-fusion
electric field waveform at all.
As to the manner of usage and operation of the present invention,
the same is apparent from the above disclosure, and accordingly, no
further discussion relative to the manner of usage and operation
need be provided.
It is apparent from the above that the present invention
accomplishes all of the objects set forth by providing new and
improved non-linear dielectrophoresis waveforms for cell fusion
which may advantageously be used to provide pre-fusion electric
field waveforms for biological cells which increase cell fusion
efficiency over biological cells treated with a constant amplitude
or a linearly increasing amplitude pre-fusion electric field
waveform. With the invention, non-linear dielectrophoresis
waveforms avoid the mechanical forces, turbulence, and heating
which result from immediately applying a high amplitude alignment
waveform to biological cells that are to undergo cell fusion. With
the invention, non-linear dielectrophoresis waveforms reduce
heating of biological cells being treated with pre-fusion electric
field waveforms for increasing cell alignment and cell membrane
contact prior to being subjected to cell fusion. With the
invention, non-linear dielectrophoresis waveforms increase cell
membrane contact between biological cells treated with pre-fusion
electric field waveforms prior to undergoing cell fusion.
Thus, while the present invention has been shown in the drawings
and fully described above with particularity and detail in
connection with what is presently deemed to be the most practical
and preferred embodiment(s) of the invention, it will be apparent
to those of ordinary skill in the art that many modifications
thereof may be made without departing from the principles and
concepts set forth herein, including, but not limited to,
variations in size, materials, shape, form, function and manner of
operation, assembly and use.
* * * * *